Abstract
Synaptic plasticity is a phenomenon underlying the ability of the central nervous system to make and retain memories. It requires the dynamic regulation of glutamate receptor expression and plasticity-related proteins. Recent work in the Williams laboratory has identified neuroprotective and plasticity-enhancing effects of the secreted amyloid precursor protein-alpha (sAPPα); positing this molecule as a potential therapeutic for Alzheimer’s Disease, a neurodegenerative disorder affecting plasticity and memory-related processes. The plasticity protein, calmodulin-dependent kinase two-alpha (CaMKIIα), has been termed the ‘molecular memory molecule’ for its crucial role in synaptic plasticity and memory, while also being dysregulated in the Alzheimer’s Disease brain. This study aimed to determine the temporal regulation of CaMKIIα by sAPPα in neurons, hypothesising that sAPPα will increase CaMKIIα activity and protein levels, reflecting an underlying mechanism of sAPPα’s plasticity-enhancing effects.
Primary hippocampal cell cultures (DIV21), prepared from Sprague Dawley rat pups (PN-0), were treated with sAPPα (1 nM; 2, 5, 10, 30 and 120-minutes). Immunocytochemistry was used to visualise and measure total CaMKIIα protein and phosphorylated protein (Thr286). Corrected total cell fluorescence (CTCF) values were generated using ImageJ, enabling the quantification of pCaMKIIα and CaMKIIα from treated cells, relative to time-matched controls. Statistical significance was determined using a linear mixed model (LMM).
We found that sAPPα temporally upregulates CaMKIIα activity (pCaMKIIα) within neuronal soma and dendrites (soma, n = 22-34 cells: 10 minutes, 1.55 fold change ± 0.86, p = 0.013; 30 minutes, 1.65 ± 1.016, p = 0.047; 120 minutes, 1.78 ± 1.15, p = 0.029; dendrites, n = 60-116 dendrites: 30 minutes, 1.65 ± 1.053, p = 0.014), while simultaneously downregulating CaMKIIα protein (soma, n = 21-29 cells: 30 minutes, 0.68 ± 0.26, p = 0.009; dendrites, n = 76-97: 5 minutes, 0.79 ± 0.47, p = 0.034; 120 minutes 0.71 ± 0.46, p = 0.003). Moreover, we provide evidence for the upregulation of microRNA, miR-142-3p, in primary cortical cells (DIV2; n = 2: 30 min, 1.59 ± 0.048, p = 0.0599) by sAPPα, suggesting the mechanism through which CaMKIIα protein expression is modulated.
These findings contribute foundational knowledge of the underlying molecular mechanisms through which sAPPα works, implicating CaMKIIα and miR-142-3p as a mechanism through which sAPPα exerts its neuroprotective and plasticity-enhancing effects. Understanding how sAPPα works at the molecular level, to regulate CaMKIIα could drive future use as a novel treatment for diseases like Alzheimer’s Disease.